The vascular endothelium, which has critical roles in maintaining blood vessel patency and integrity, regulates the state of contraction of the underlying layer of smooth muscle. Recently, an endothelium-derived peptide, endothelin 1 (ET1), with potent vasoconstrictor and mitogenic properties was identified. Subsequently, genes encoding two related vasoconstrictor peptides, endothelins 2 and 3 (ET2 and ET3), were reported. The overall objective of the proposed research is to investigate the roles of this family of endothelin peptides in normal physiology and in the pathogeneses of cardiovascular disorders including atherosclerosis and systemic and pulmonary hypertension. To understand the biological functions of the endothelins, it is necessary to identify the cells and tissues producing these vasoactive peptides. Preliminary evidence suggests that the ET1 and ET3 genes are coexpressed in human fetal lung, spleen, and pancreas. Abundant ET1 mRNA is present in endothelial cells, while ET3 mRNA was not detected. ET2 gene expression appears more limited and was detected in a single pulmonary cell line. The specific aims of the proposed research include further investigation of the tissue-distribution and developmental regulation of endothelin gene expression using in situ hybridization and RNA blot hybridization techniques. Endothelin-specific antisera will be developed and employed to identify cell types synthesizing endothelins. Regulation of production of the three endothelins in these cells will be investigated and compared. Metabolic labeling experiments will be performed to study post- translational processing of endothelin precursors and cellular mechanisms of endothelin secretion, potentially identifying sites for future pharmacologic intervention in the maturation of endothelin peptides. Polarity of ET1 secretion by endothelial cells will be studied, and cellular mechanisms of polarized secretion in these cells will be explored. Identification of the cells producing endothelins and mechanisms regulating their production and secretion will provide important insights into the physiological roles of this family of vasoactive peptides. Because it is highly likely that one or more member of the endothelin gene family participates in the pathogenesis of cardiovascular disorders, investigations of the biosynthesis and secretion of these vasoactive peptides may have important therapeutic implications.